Mechanism underlying ALS/FTD C9orf72 DPR-induced cytotoxicity

• 1、Department of Pharmacology, Soochow University, Suzhou, 215123

Abstract：Objective: To explore the cytotoxicmechanismof five dipeptide-repeat proteins (DPRs: PR, GR, GA, GP,PA) translatedfrom the non-coding region of amyotrophic lateral sclerosisand frontotemporal dementia-related gene C9orf72. Methods: DPRs were overexpressed in HEK293 cells. The expression and localization of DPRs were determined by cell biology and biochemical experiments. The influence of DPRs on cytotoxicity were explored byPI stainingexperiment. Proteasomal inhibitor MG132 and autophagy inhibitor BafiA1 were used to detect the responses of DPRs at thesubcellular locationand protein level. To explore the relationship between DPRs and membrane-less ormembrane-bound organelles, we co-labelledDPRs, nucleolus and endoplasmic reticulum. Finally,in vitrophase separationand intracellular fluorescence recovery after photo-bleaching were conducted to investigate whether arginine-rich DPRs can undergo phase separation and its effect on nucleolar functions. Results: Arginine-rich DPRscould form membrane-less condensates in the nuclei and co-localized with the GC markerof nucleolus. PR and GR were more cytotoxic than the other DPRs, and were not highly regulatedby the ubiquitin-proteasome system or autophagy. PR and GR could phase separatein vivo or in vitroand form fibers in vitro, inhibit the fluidity of nucleoli in cellsand affect nucleolar functions and then cause cytotoxicity and promote cell death.

Keywords： Neurodegenerative diseases amyotrophic lateral sclerosis frontotemporal dementia C9orf72 dipeptide-repeat proteins